Sesquiterpenoid derivatives having adipocyte differentiation inhibitory effect

ABSTRACT

An inhibitor of the rennin-angiotensin system is useful for the treatment or prevetion of the lipodystrophy syndrome, e.g. in AIDS patients also receiving anti-retroviral therapy.

TECHNICAL FIELD

The present invention relates to adipocyte differentiation inhibitorscontaining sesquiterpenoid derivatives that have an inhibitory effect onadipocyte differentiation as active ingredients, and pharmaceuticalcompositions and food compositions for prevention, improvement, ortreatment of diseases caused by the induction of adipocytedifferentiation.

BACKGROUND ART

Obesity is caused by overeating, lack of exercise, abnormal feedingpattern, genetic predisposition, thermogenesis impairment, and such.Excess energy resulting from imbalance of intake and expenditure ofenergy is stored in adipocytes, and these adipocytes gather to formadipose tissue. That is, obesity is a condition of hyperplasia ofadipose tissues.

In addition, obesity is thought to be a major risk factor for heartdiseases (angina pectoris, myocardial infarction, cardiac hypertrophy,heart failure, etc.), vascular disorders (hypertension,arteriosclerosis, cerebral thrombosis, cerebral infarction, etc.)diabetes, gout, hyperlipemia, fatty liver, gallstone, pancreatitis,osteoarthritis, hernia, and such.

Adipocytes are produced from mesenchymal cells and are formed bydifferentiation induction from adipocyte precursors. Therefore,adipocyte formation can be suppressed by inhibiting differentiationinduction of adipocyte precursors to form adipocytes. When the formationof adipocyte is suppressed, adipose tissue will not be formed. Thus,compounds that inhibit differentiation of adipocytes are considered tohave preventive or therapeutic effects against obesity related diseases.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to identify compounds havinginhibitory effects on adipocyte differentiation, and to providepharmaceutical compositions and food compositions for prevention,improvement, or treatment of obesity or obesity related diseases.

In order to achieve the above-mentioned objective, the present inventorsmade every effort to study natural products that inhibit thedifferentiation induction of adipocyte precursors to adipocytes. As aresult, sesquiterpenoid derivatives extracted from a Compositae plant,Calea, growing in Central and South America, were found for the firsttime to possess an excellent inhibitory effect on the induction ofadipocyte differentiation. Therefore, there is a great expectation thatthese sesquiterpenoid derivatives may be used in pharmaceuticalcompositions and food compositions for prevention, improvement, ortreatment of obesity or obesity related diseases.

More specifically, the present invention relates to an adipocytedifferentiation inhibitor containing a sesquiterpenoid derivative thathas an inhibitory effect on adipocyte differentiation as an activeingredient, and also to pharmaceutical compositions and foodcompositions for prevention, improvement, or treatment of obesity orobesity related diseases, and more specifically provides:

[1] a Calea extract having an inhibitory effect on adipocytedifferentiation;

[2] the Calea extract according to [1], wherein said Calea is selectedfrom the group consisting of Calea urticifolia, Calea pinnatifida, Caleauniflora, Calea axillaris, Calea insignis, Calea integrifolia, Caleanelsonii, Calea peduncularis, Calea pringlei, Calea purpusii, Caleasabazioides, Calea savannarum, Calea scabra, Calea sororia, Caleastandleyi, Calea tejadae, and Calea zacatechichi;

[3] a sesquiterpenoid derivative having an inhibitory effect onadipocyte differentiation;

[4] a sesquiterpenoid derivative of any one of structural formula (1) to(4):

[5] a sesquiterpenoid derivative having an inhibitory effect onadipocyte differentiation of any one of structural formula (1) to (4) of[4] and (5) to (7):

[6] an adipocyte differentiation inhibitor containing the Calea extractaccording to [1] or [2], or the sesquiterpenoid derivative according to[3] or [5] as an active ingredient;

[7] a pharmaceutical composition for prevention, improvement, ortreatment of obesity or obesity related diseases, containing the Caleaextract according to [1] or [2], or the sesquiterpenoid derivativeaccording to [3] or [5] as an active ingredient;

[8] the pharmaceutical composition according to [7], wherein the diseaseis selected from the group consisting of heart disease, vasculardisorder, diabetes, gout, hyperlipemia, fatty liver, gallstone,pancreatitis, osteoarthritis, and hernia; and

[9] a food composition forprevention or improvement of obesity orobesity related diseases, containing the Calea extract according to [1]or [2], or the sesquiterpenoid derivative according to [3] or [5] as anactive ingredient.

The present inventors discovered that the Calea plant extract,sesquiterpenoid derivatives, have an inhibitory effect on the inductionof adipocyte differentiation. Therefore, the present invention providesCalea extract having adipocyte differentiation inhibitory effect.

Calea is a plant belonging to Compositae and grows mainly in Central andSouth America. The Calea of the present invention includes Caleaurticifolia, Calea pinnatifida, Calea uniflora, Calea axillaris, Caleainsignis, Calea integrifolia, Calea nelsonii, Calea peduncularis, Caleapringlei, Calea purpusii, Calea sabazioides, Calea savannarum, Caleascabra, Calea sororia, Calea standleyi, Calea tejadae, and Caleazacatechichi, but is not particularly limited to these species orvariations.

The term “Calea extract” herein refers to a substance extracted from allor a portion of the Calea plant. The Calea extract of the presentinvention is typically in a liquid form (for example, Calea extractsolution) but is not limited thereto, and includes also powderedsubstances made upon drying the extracted solution, or substanceobtained by cutting or grinding an arbitrary portion of Calea. The Caleaextract of the present invention can be obtained by extracting all or anarbitrary portion of the Calea plant. However, extract from the entireplant is especially preferable.

The Calea extract of the present invention can be obtained according tothe method for preparing plant extract solutions generally performed bythose skilled in the art. For example, the Calea extract solution can beobtained according to the following method. First, the collected plantis cut and is dried in a cool place away from direct sunlight. Then,following the production of powder using a grinder, a variety ofappropriate organic solvents, water, or their mixed solutions are usedfor extraction by heated reflux. Next, the extracted solution isfiltered, and concentrated under reduced pressure to obtain aconcentrated extract.

Whether the Calea extract obtained by the above-mentioned method has aninhibitory effect on adipocyte differentiation or not can be evaluated,for example, by measuring acetic acid (¹⁴C—CH₃COOH) incorporationactivity, or by glucose (¹⁴C-2-deoxyglucose) incorporation activitydescribed later in the Examples. Examples of biochemical evaluationmethods include measurements of glucose-6-phosphate dehydrogenaseactivity, neutral fat releasing activity, and such. In addition,regarding detection at the mRNA level, evaluation can be conducted bydetecting adipocyte differentiation markers, such as the aP2 gene.

Furthermore, the present inventors discovered that sesquiterpenoidderivatives contained in Calea extract have an inhibitory effect onadipocyte differentiation. Therefore, the present invention providessesquiterpenoid derivatives having adipocyte differentiation inhibitoryeffect.

The sesquiterpenoid derivative of the present invention having adipocytedifferentiation inhibitory effect may be a natural compound (forexample, a compound extracted from plants other than Calea) or anartificially synthesized compound.

Compounds having the characteristics (chemical formula, structuralformula, molecular weight, etc.) indicated in the following (1) to (7)may be cited as examples of the sesquiterpenoid derivatives of thepresent invention having adipocyte differentiation inhibitory effects.

The above-mentioned compounds 1, 4, 7, and 12 are compounds found forthe first time in the present invention. The present invention alsoprovides these novel compounds. Furthermore, the sesquiterpenoidderivative of the present invention includes analogs of theabove-mentioned compounds, or compounds produced as derivatives based onthese compounds so long as they possess an inhibitory effect onadipocyte differentiation. For example, a compound wherein a functionalgroup of the above-mentioned compound is replaced with anotherfunctional group is included in the compound of the present invention solong as it has an inhibitory effect on adipocyte differentiation.Furthermore, some compounds are known to become pro-drugs (a drug thatshows activity only after being metabolized in vivo due to modificationof the chemical structure) by esterification. Such chemically modifiedsesquiterpenoid derivatives (such as pro-drug, etc.) are also includedin the present invention.

In addition, the present invention provides Calea extract havingadipocyte differentiation inhibitory effect, or adipocytedifferentiation inhibitor containing sesquiterpenoid derivatives as theactive ingredient. Adipocytes are produced from mesenchymal cells andare formed by differentiation induction from adipocyte precursors.Compounds having an inhibitory effect on adipocyte differentiationsuppress the formation of adipocytes, and as a result, adipose tissueformation is expected to be suppressed. Therefore, such compounds areexpected to be not only useful as reagents for suppressing adipocytedifferentiation, but also effective in prevention, improvement, ortreatment of obesity or obesity related diseases. Thus, the presentinvention provides Calea extract, or pharmaceutical compositionscontaining sesquiterpenoid derivatives as the active ingredient forprevention, improvement, or treatment of obesity or obesity relateddiseases.

The term “prevention” herein includes not only prevention before havingthe disease, but also prevention against recurrence of the disease aftertreatment. Specifically, “obesity related diseases” that are the targetsof prevention, improvement, or treatment of the present inventioninclude, for example, heart diseases (angina pectoris, myocardialinfarction, cardiac hypertrophy, heart failure, etc.) , vasculardisorders (hypertension, arteriosclerosis, cerebral thrombosis, cerebralinfarction, etc.), diabetes, gout, hyperlipemia, fatty liver, gallstone,pancreatitis, osteoarthritis, hernia, and such, but are not particularlylimited to these diseases.

When using the adipocyte differentiation inhibitor, the anti-obesitydrug for prevention, improvement, or treatment of obesity, and thepharmaceutical compositions for prevention, improvement, or treatment ofobesity related diseases of the present invention, it is prepared as ageneric medical formulation. For example, the medicament of the presentinvention is prescribed in a form suited for oral administration orparenteral administration as a formulation, such as pharmaceuticalcompositions or tablet, pill, powder, granule, encapsulated formulation,troche, syrup, solution, emulsion, suspension, injection obtained bymixing with carriers (excipient, binder, disintegrant, corrigent fortaste, corrigent for smell, emulsifier, diluent, adjuvant, etc.)acceptable for formulation.

Examples of excipient include lactose, cornstarch, white sugar, glucose,sorbitol, plasma cellulose, and such. Examples of binders includepolyvinyl gum arabia, tragacanth, gelatin, shellac,hydroxypropylcellulose, hydroxypropyl starch, polyvinylpyrrolidone, andsuch.

Examples of disintegrants include starch, agar, gelatin powder,crystalline cellulose, calcium carbonate, sodium bicarbonate, calciumcitrate, dextran, pectin, and such. Examples of lubricants are magnesiumstearate, talc, polyethylene glycol, silica, hardened vegetable oil, andsuch. Further, coloring agents acceptable for addition into medicamentsmay be used. Example of corrigents for taste and smell include cocoapowder, 1-menthol, aromatic acids, peppermint oil, Borneo camphor,cinnamon powder, and such. These tablets and granules may beappropriately coated as necessary with sugar coating, gelatin coating,and so on.

When preparing an injection, pH regulator, buffer, stabilizer,preservative, and such, are added as necessary, and is made into asubcutaneous, intramuscular, or intravenous injection by conventionalmethods. The injection can be a formulation that is prepared just beforeuse by storing the solution into a container, then producing a solidformulation by freeze drying and such. A single dose can be stored intoa container, and a dose can be stored into the same container.

The dosage of the adipocyte differentiation inhibitor, the anti-obesitydrug for prevention, improvement, or treatment of obesity, and thepharmaceutical composition for prevention, improvement, or treatment ofobesity related diseases of the present invention is determined byconsidering the type of dosage form, administration method, age andweight of the subject (mammals including humans), condition of thesubject, and such. Specifically, for an adult patient, for example, 0.01to 600 mg can be administered orally per day as 1 to several doses.Examples of doses are more preferably 0.1 to 400 mg/day, and even morepreferably 1 to 200 mg/day. Although these doses vary depending on theweight and age of the patient, and on the administration method, oneskilled in the art can appropriately select the correct dose. It is alsopreferable to appropriately determine the administration perioddepending on the healing course of the patient, and such.

A preferred embodiment of the above-mentioned pharmaceutical compositionof the present invention may be an oral composition. Specifically, theoral composition may be, for example, in a dry powdered form of theCalea extract of the present invention. The dry powder may be prepared,for example, according to the method selected from (a) and (b) mentionedbelow:

-   (a) freeze-drying method: The extract solution is suspended in    water, and frozen. Then, the frozen solution is dried by subliming    water by reduced pressure to powderize the extract solution; and-   (b) spray-drying method: The extract solution or suspension is    sprayed into hot air and is dried instantaneously to obtain a    spherical dried substance.

In addition, the present invention provides food compositions forprevention or improvement of obesity or obesity related diseases thatcontain Calea extract, or sesquiterpenoid derivatives as activeingredients. Examples of the food compositions of the present inventionare health food, functional food, specified health food, nutritionalsupplements, enteral nutrition, and such, but are not limited to thesefoods as long as it has the effect of preventing or improving obesity orobesity related diseases. The food compositions of the present inventionmay be used favorably in the form of the above-mentioned oralcomposition. Further, the present invention includes the use of thedried powder or the extract for producing oral compositions forimprovement, or prevention, or otherwise prevention of recurrence ofdiseases caused by adipocyte differentiation induction in mammals,including humans. The production method of the oral compositions is awell known, frequently used technology for those skilled in the art.More specifically, the sesquiterpenoid derivative of the presentinvention or Calea extract solution or dried Calea powder containing thesesquiterpenoid derivative can be processed into health food, functionalfood, specified health food, nutritional supplements, enteral nutrition,and such, by mixing compositions that are acceptable in terms of foodsanitation. For example, compositions, such as stabilizers,preservatives, coloring agents, fragrances, and vitamins, can be addedappropriately to the above-mentioned sesquiterpenoid derivatives, or tothe Calea extract solution and dried Calea powder containing thesesquiterpenoid derivatives, mixed, and then formed appropriately byconventional methods for oral compositions, such as tablet, particulate,granule, powder, capsule, liquid, cream, beverage, and such.

Any patents, patent applications, and publications cited herein areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the inhibitory effect of Calea extract onadipocyte differentiation induction. The ordinate shows ¹⁴C—CH₃COOHincorporation (cpm). “−Ins.”, “+Ins.”, and “Ins.+” on the abscissaindicate no addition of insulin, addition of insulin, and addition ofboth insulin and Calea extract to the sample, respectively.

FIG. 2 is a diagram showing the inhibitory effect of Calea extract onadipocyte differentiation induction. The ordinate shows¹⁴C-2-deoxyglucose incorporation (cpm). “−Ins.”, “+Ins.”, and “Ins.+” onthe abscissa indicate no addition of insulin, addition of insulin, andaddition of both insulin and Calea extract to the sample, respectively.

FIG. 3 is a diagram showing the isolation process of compounds(sesquiterpenoid derivatives) having an inhibitory effect on adipocytedifferentiation induction.

FIG. 4 is a diagram showing the inhibitory effect of sesquiterpenoidderivatives on adipocyte differentiation induction. The ordinate shows¹⁴C—CH₃COOH incorporation (cpm/mg protein) “−Ins.”, and “+Ins.” on theabscissa indicate no addition of insulin, and addition of insulin to thesample, respectively. 1, 3, 4, 6, 7, 11, and 12 on the abscissa indicatesamples to which compounds 1, 3, 4, 6, 7, 11, and 12, respectively, havebeen added in combination with insulin.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specificallyby way of Examples, but the present invention is not limited to theseExamples.

EXAMPLE 1 Evaluation of the Inhibitory Effect of Calea Extract Solutionon Adipocyte Differentiation Induction

Whether Calea extract solution has an inhibitory activity on adipocytedifferentiation induced by the addition of insulin was investigated.First, the aerial portion of Calea (170 g) was ground, and extracted byheated reflux using acetone (24 hours×2 times) to produce Calea extractsolution. The extracted solution was filtered, and then concentratedunder reduced pressure to yield a concentrated extract. Mouse adipocyteprecursors (3T3-L1) obtained from Human Science Research Resources Bankwas used as the adipocyte in the present experiment. Normal successivecultivation was performed using Dulbecco's modified Eagle MEM mediumcontaining 10% calf serum (CS) at 37° C. in the presence of 5% CO₂. Toevaluate the inhibitory effect on differentiation induction intoadipocytes, the above-mentioned cells were removed from a dish bytrypsin treatment to obtain cell suspension, and then after separationby centrifugation at 1,000 rpm for 3 minutes at 4° C., the concentrationwas adjusted to 3.5×10⁴ cells/ml, and 200 μl aliquots were placed into a96-well plate to prepare an examination plate. After 72 hours, 2 μl eachof a sample dissolved in 10% DMSO was placed into each of theexamination plate wells. Simultaneously, dexamethasone andisobutylmethyl xanthine were added to a final concentration of 20 μM and10 mM, respectively. After 48 hours, the media was removed, 200 μl offresh media was added, and the same concentration of sample was addedagain. Furthermore, insulin was added to a final concentration of 17 μM.Subsequently, media exchange and addition of accompanying reagents andinsulin were carried out at 3-day intervals. 8 to 10 days later, mediawere removed, cells were washed with PBS(−), immobilized using 10%formalin solution, adipose granules stored in the cells were stainedwith Oil Red-O/60% isopropanol saturated solution for 1 hour at roomtemperature. After staining, excess Oil Red O solution was removed usingPBS(−). Observation of the stained adipose granules under a lightmicroscope confirmed that the cells had undergone differentiationinduction.

In a system for evaluating the inhibitory activity against adipocytedifferentiation induced by insulin addition, acetic acid (¹⁴C—CH₃COOH)incorporation activity, which is used as an index for differentiationinduction activity, and incorporation activity of glucose(¹⁴C-2-deoxyglucose), which is known not to be metabolized in cells,were used as indices.

In the system where acetic acid incorporation activity was used as anindex, first the cells were placed into a 24-well plate at a density of4.2×10⁴ cell/well. After 72 hours, Calea extract solution, IBMX (10 mM),and dexamethazone (20 μM) were added, and was cultivated for another 48hours. Then, the media was exchanged to a fresh media, and Calea extractsolution and insulin (17 μM) were added. The media was exchanged to afresh media at 3-day intervals, and Calea extract solution and insulinwere added each time. After 9 days of cultivation following insulinaddition, 14C-sodium acetate was added to a concentration of 1 μCi/ml,and was cultivated for 3 hours. Then, the media was removed, the cellswere washed twice using 2 ml of PBS(−) and then dried for a few minuteson a clean bench. Next 1 ml isopropanol was gently added, left standingfor a few minutes, and then isopropanol was collected and transferred toa scintillation vial. Isopropanol extraction was performed 2 more times.Using a liquid scintillation counter, radioactivity of the isopropanolfraction (adipose component) was measured. Next, upon completely dryingthe isopropanol extracted cells, the cells were dissolved using 200 μlof 0.1 N NaOH, and then were neutralized with 200 μl of 1 M Tris-HClpH7.5. Then, protein quantification was performed according to theBradford dye-binding method. For the adipose synthesis activity,¹⁴C—CH₃COOH incorporation activity per amount of protein was used as anindex. Upon analysis, approximately 50% and nearly 100% incorporationinhibitory activities were indicated at concentrations of 4 μg/ml and 8μg/ml extract solutions, respectively (FIG. 1).

On the other hand, in a system wherein glucose incorporation activity isused as an index, cells were placed into a 24-well plate at a density of4.2×10⁴ cells/well. After 72 hours, Calea extract solution, IBMX (10mM), and dexamethazone (20 μM) were added, and cells were cultivated foranother 48 hours. Then, the media was exchanged to a fresh media, andCalea extract solution and insulin (17 μM) were added. The media wasexchanged to a fresh media at 3-day intervals, and Calea extractsolution and insulin were added each time. After 9 days of cultivationafter insulin addition, the media was replaced with 500 μl/well ofglucose deficient DMEM media, and was cultivated for 15 minutes. Then,¹⁴C-2-deoxyglucose (Amersham Pharmacia Biotech) was added to a finalconcentration of 0.25 μCi/ml, and was cultivated for 15 minutes. Then,the cells were washed three times with ice cold PBS(−) containing 10 mMglucose, were removed from the dish using trypsin, and were transferredto a scintillation vial. Using a liquid scintillation counter,radioactivity of ¹⁴C-2-deoxyglucose incorporated into the cells wasmeasured. Upon analysis, approximately 25% and approximately 15% ofincorporation inhibitory activities were indicated at extract solutionconcentrations of 4 μg/ml and 8 μg/ml, respectively (FIG. 2).

EXAMPLE 2 Identification of Compounds having Adipocyte DifferentiationInduction Inhibitory Activity in Calea Extract Solution

From Example 1, a compound existing in the Calea extract solution wasthought to inhibit differentiation induction of adipocytes.Consequently, compounds having adipocyte differentiation inductioninhibitory activity were identified.

Specifically, first, the above-mentioned compound was isolated by theprocess indicated in FIG. 3. The aerial portion of Calea (170 g) wasground, and was extracted by heated reflux (24 hours×2 times) usingacetone. After concentrating the extracted solution under reducedpressure (9.8 g), the solution was distributed into chloroform andwater. Upon concentrating the chloroform fraction under reduced pressure(7.1 g) , the concentrated extract was dissolved in CHCl₃, and waschromatographed through a silica gel column (50×200 mm: 400 ml).Practically, 1200 ml each of solvents composed of CHCl₃:MeOH=30:1and20:1were used for elution in order. 6fractions, each containing 400 ml,were obtained. Active fraction 2 was concentrated under reduced pressure(2.1 g). Next, the concentrated extract of the active fraction wasdissolved in 5 ml of MeOH, and was chromatographed on a reverse-phasesilica gel column (38×130 mm: 150 ml Cosmosil 140C18-OPEN (Nakarai)).Specifically, the sample was added to a column material equilibratedwith 40% MeOH, and was eluted using 40 and 65% MeOH in order. The activefraction eluted with 65% MeOH was concentrated under reduced pressure,and 1.14 g of active extract was obtained. Furthermore, usingreverse-phase HPLC, the active ingredient was separated. Upon analysis,7 compounds having adipocyte differentiation induction inhibitoryactivity were isolated under the following conditions: column, MightysilRP-18 20×250 mm; column temperature, 40° C.; flow rate, 11.342ml/minute; detector, 210 nm; solvent, 42% CH₃CN. Following theconcentration under reduced pressure, each compound was recrystallizedfrom Et₂O. The retention times of the isolated compounds were, compound1: 13.5 minutes; compound 3: 18.1 minutes; compound 4: 20.2 minutes;compound 6: 23.3 minutes; compound 7: 25.3 minutes; compound 11: 35.1minutes; and compound 12: 38.3 minutes.

Next, to identify the above-mentioned compounds, their physico chemicalproperties were investigated (Table 1). TABLE 1 1 3 4 + 5 6 7 11 12m.p.(° C.) 142-144 133-135 170-172 150-153 99-101 decomp. 98-100 EI/MS406 406 450 432 434 cation FAB/MS 433 449 [α]₀ ²⁵ (c 0.001, CHCl₃) 184.2192.2 92.1 154 168.7 — 195.4

Furthermore, by ¹H (Table 2, Table 3) and ¹³C-NMR analysis (Table 4), aswell as various two-dimensional NMR, relative configuration of eachcompound was determined and respective structural formula wasdetermined. As a result, all of these compounds having the inhibitoryactivity on the induction of adipocyte differentiation were revealed tobe sesquiterpenoid derivatives. In particular, compounds 1, 4, 7, and 12were novel sesquiterpenoid derivatives. TABLE 2 1 3 4 6 7  2 4.21(d,4.1Hz, 1H) 6.50(d, 12.0, 1H) 3.08(dd, 16.0, 8.0Hz, 1H) 4.30(d, 4.4Hz,1H) 4.27(d, 4.1Hz, 1H) 3.60(dd, 16.0, 9.5Hz, 1H)  3 3.30(dd, 9.4, 4.3Hz,1H) 5.98(dd, 12.0 and 12.0, 1H) 5.92(m, 1H) 3.34(dd, 9.4, 4.3Hz, 1H)3.35(dd, 9.4, 4.1Hz, 1H)  4 1.45:assigned from 3.05(m, 1H) 1.45:assignedfrom 1.45:assigned from H—H COSY H—H COSY H—H COSY  5 1.46, 1.90(each m,1H) 1.38(m, 1H) 2.77(dd, 15.0, 4.0Hz, 1H) 1.50, 1.96(each m, 1H) 1.51,1.95(each m, 1H) 1.80(m, 1H) 2.86(dd, 15.0, 3.6Hz, 1H)  6 4.80(dd, 12.2,4.5Hz, 4.55(dd, 11.7, 4.9Hz, 1H) 4.95(m, 1H) 4.84(dd, 12.1, 4.5Hz, 1H)4.86(dd, 12.1, 4.3Hz, 1H) 1H)  7 2.34(br s, 1H) 2.60(s, 1H) 2.68(m, 1H)2.38(br s, 1H) 2.39(br s, 1H)  8 5.66(dd, 10, 1.7Hz, 1H) 5.55(s, 1H)5.90(dd, 10.5, 1.5Hz, 1H) 5.75(dd, 9.7, 2.0Hz, 1H) 5.72(dd, 9.9, 1.7Hz,1H)  9 5.73(d, 10Hz, 1H) 5.55(s, 1H) 5.70(d, 8.1Hz, 1H) 5.85(d, 12.0Hz,1H) 5.82(d, 9.9Hz, 1H) 13 5.81, 6.31(each m, 1H) 5.80, 6.25(each d, 1.1,1H) 5.72(s, 1H) 5.84, 6.34(each s, 1H) 5.85(d, 1.0Hz, 1H) 6.30(d, 3.4Hz,1H) 6.34(s, 1H) 14 1.19(d, 7.1Hz, 3H) 1.05(d, 6.3, 3H) 1.86(s, 3H)1.23(d, 6.1Hz, 3H) 1.21(d, 6.1Hz, 3H) 15 1.45(s, 3H) 1.30(s, 3H) 1.35(s,3H) 1.46(s, 3H) Acetyl  2′ 1.95(s, 3H) Meacr  3′ 5.56, 6.03(each m, 1H)5.45, 5.95(each s, 1H) 5.60, 6.00(each m, 1H) 5.51(m, 1H) 5.58(m, 1H)5.97(s, 1H) 6.05(s, 1H)  4′ 1.83(br s, 1H) 1.78(s, 3H) 1.87(s, 3H)1.78(s, 3H) 1.83(s, 3H) Acetyl  2″ 2.03(s, 3H) 2.06(s, 3H) Meacr  3″5.66(m, 1H) 6.16(s, 1H)  4″ 1.90(s, 3H) iBu  2″ 2.57(dq, 7.1, 7.1Hz, 1H) 3″ 1.10(d, 7.1Hz, 3H)  4″ 1.16(d, 7.1Hz, 3H) 10-OH 3.89(br s, 1H)4.28(br s, 1H) 4.05(br s, 1H)

TABLE 3 11 12 12 in d 6-benzene 6.03(dd, 12.0, 11.5Hz, 1H) 5.95(dd,12.0, 11.5Hz, 1H) 4.95(dd, 11.8, 11.5Hz, 1H) 6.64(d, 12.0Hz, 1H) 6.54(d,12.0Hz, 1H) 6.02(d, 11.8Hz, 1H) 3.13(m, 1H) 3.06(m, 1H) 2.80(m, 1H)1.45, 1.83(each m, 1H) 1.38, 1.79(each m, 1H) 0.95, 1.25(each m, 1H)4.63(dd, 12.0, 4.9Hz, 1H) 4.55(dd, 11.7, 4.5Hz, 1H) 4.38(dd, 11.7,4.9Hz, 1H) 2.66(s, 1H) 2.56(s, 1H) 2.48(br s, 1H) 5.69(s, 1H) 5.58(s,1H) 6.00(dd, 9.8, 2.1Hz, 1H)) 5.69(s, 1H) 5.58(s, 1H) 5.73(d, 9.8Hz, 1H)5.84(d, 1.2Hz, 1H) 5.76(d, 1.2Hz, 1H) 5.25(d, 1.5Hz, 1H) 6.33(s, 1H)6.25(br s, 1H) 6.18(d, 1.0Hz, 1H) 1.15(d, 6.3Hz, 3H) 1.08(d, 7.1Hz, 3H)0.58(d, 6.3Hz, 3H) 1.34(s, 3H) 1.25(s, 3H) 1.09(s, 3H) 5.61(br s, 1H)5.47(br s, 1H) 5.19(m, 1H) 6.13(s, 1H) 5.95(s, 1H) 6.23(br s, 1H)1.88(s, 3H) 1.74(s, 3H) 1.83(br s, 3H) 5.48(br s, 1H) 5.94(s, 1H)1.76(s, 3H) 2.46(dq, 7.1, 7.1Hz, 1H) 2.34(dq, 7.1, 7.1Hz, 1H) 1.01(d,7.1Hz, 3H) 0.98(d, 7.1Hz, 3H) 1.08(d, 7.1Hz, 3H) 0.98(d, 7.1Hz, 3H)4.13(br s, 1H) 4.04(br s, 1H)

TABLE 4 12 in d 1 3 4 6 7 11 12 6-benzene C-1 206.0 204.7 210.7 206.1206.0 204.9 205.1 204.9  2 55.6 125.3 36.0 55.7 55.7 148.1 148.4 148.2 3 62.8 148.2 121.5 62.8 62.7 125.4 125.7 125.5  4 26.0 28.3 136.6 26.025.9 28.3 28.6 28.3  5 38.8 40.2 35.9 38.8 38.7 40.3 40.6 40.5  6 74.676.3 76.6 74.7 74.7 76.4 76.4 76.2  7 40.9 41.2 42.0 40.9 40.9 41.2 41.741.9  8 73.9 74.4 67.0 73.8 73.8 74.3 74.3 75.1  9 71.5 73.8 72.5 71.770.8 74.0 73.2 73.9 10 79.7 79.2 80.3 79.9 79.8 79.4 79.7 79.8 11 134.3134.5 134.1 134.3 134.2 134.6 134.9 135.6 12 168.3 168.7 168.1 168.4168.0 168.8 169.1 168.6 13 126.8 126.6 124.4 126.7 126.8 126.5 127.0126.0 14 18.6 19.7 25.4 18.6 18.5 19.7 20.0 19.6 15 24.5 23.4 22.3 24.624.4 23.6 23.9 23.8  1′ 165.2 170.3 165.2 165.4 165.0 166.6 165.6 165.8 2′ 134.8 20.3 135.1 134.8 134.8 134.7 135.2 135.9  3′ 127.3 126.9 127.0127.3 127.8 127.6 127.4  4′ 18.1 18.1 18.0 18.0 18.0 18.4 18.4  1″ 170.4165.3 170.4 166.7 176.0 165.4 176.8 176.6  2″ 20.2 134.9 20.4 134.5 33.9134.9 34.3 34.4  3″ 127.1 128.2 18.6 126.9 19.0 19.0  4″ 18.0 18.0 18.818.1 19.3 19.3

EXAMPLE 3 Examination of the Relationship Between Structure and Activityof Calea Extract

Structure-activity relationships of the 7 compounds obtained in Example2 were examined. Inhibitory activities of respective compounds on thedifferentiation induction of 3T3-L1 cells into adipocytes were comparedin the system described in Example 1 that uses acetic acid incorporationactivity as the index. As a result, high incorporation inhibitoryactivity was confirmed for compounds 11 and 6. Ranking the compounds inthe order of higher inhibitory activity, the order was: compound11>6>4>3=12>1>7 (FIG. 4).

Industrial Applicability

The present invention provides sesquiterpenoid derivatives havingadipocyte differentiation inhibitory effect. Furthermore, the presentinvention provides pharmaceutical compositions and food compositions forprevention, improvement, or treatment of obesity or obesity relateddiseases that contain Calea extract or the sesquiterpenoid derivative asan active ingredient. The use of these compositions presents greatexpectations for effective prevention, improvement, or treatment ofobesity or obesity related diseases.

1. A Calea extract having an inhibitory effect on adipocytedifferentiation.
 2. The Calea extract according to claim 1, wherein saidCalea is selected from the group consisting of Calea urticifolia, Caleapinnatifida, Calea uniflora, Calea axillaris, Calea insignis, Caleaintegrifolia, Calea nelsonii, Calea peduncularis, Calea pringlei, Caleapurpusii, Calea sabazioides, Calea savannarum, Calea scabra, Caleasororia, Calea standleyi, Calea tejadae, and Calea zacatechichi.
 3. Asequiterpenoid derivative having an inhibitory effect on adipocytedifferentation.
 4. A sequiterpenoid derivative of any one of structuralformulae (1) to (4):


5. The sesquiterpenoid derivative, according to claim 3, having any oneof structural formulae (1) to (7):


6. An adipocyte differentiation inhibitor comprising, as an activeingredient one or more of the following: a) a Calea extract having aninhibitory effect on adipocyte differentiation; and b) a sesquiterpenoidderivative having an inhibitory effect on adipocyte differentiation. 7.A pharmaceutical composition for prevention, improvement, or treatmentof obesity or obesity related diseases, comprising as an activeingredient one or more of the following: a) a Calea extract having aninhibitory effect on adipocyte differentiation; and b) a sesquiterpenoidderivative having an inhibitory effect on adipocyte differentiation. 8(cancel).
 9. A food composition for prevention or improvement of obesityor obesity related diseases, as comprising as an active ingredient oneor more of the following: a) a Calea extract having an inhibitory effecton adipocyte differentiation; and b) a sesquiterpenoid derivative havingan inhibitory effect on adipocyte differentiation.
 10. A method for theprevention, improvement, or treatment of obesity, or an obesity relateddisease, wherein said method comprises administering, to a patient inneed of such prevention, improvement, or treatment, an effective amountof one or more of the following: a) a Calea extract having an inhibitoryeffect on adipocyte differentiation; and b) a sesquiterpenoid derivativehaving an inhibitory effect on adipocyte differentiation.
 11. Themethod, according to claim 10, wherein the disease is selected from thegroup consisting of heart disease, vascular disorders, diabetes, gout,hyperlipemia, fatty liver, gallstone, pancreatitis, osteoarthritis, andhernia.
 12. The method, according to claim 10, wherein said Calea isselected from the group consisting of Calea urticifolia, Caleapinnatifida, Calea uniflora, Calea axillaris, Calea insignis, Caleaintegrifolia, Calea nelsonii, Calea peduncularis, Calea pringlei, Caleapurpusii, Calea sabazioides, Calea savannarum, Calea scabra, Caleasororia, Calea standleyi, Calea tejadae, and Calea zacatechichi.
 13. Themethod, according to claim 10, which comprises administering a compoundhaving any one of structural formulae (1) to (7):


14. The adipocyte differentiation inhibitor, according to claim 6,wherein said Calea is selected from the group consisting of Caleaurticifolia, Calea pinnatifida, Calea uniflora, Calea axillaris, Caleainsignis, Calea integrifolia, Calea nelsonii, Calea peduncularis, Caleapringlei, Calea purpusii, Calea sabazioides, Calea savannarum, Caleascabra, Calea sororia, Calea standleyi, Calea tejadae, and Caleazacatechichi.
 15. The adipocyte differentiation inhibitor, according toclaim 6, having any one of structural formulae (1) to (7):

16 The pharmaceutical composition, according to claim 7, wherein saidCalea is selected from the group consisting of Calea urticifolia, Caleapinnatifida, Calea uniflora, Calea axillaris, Calea insignis, Caleaintegrifolia, Calea nelsonii, Calea peduncularis, Calea pringlei, Caleapurpusii, Calea sabazioides, Calea savannarum, Calea scabra, Caleasororia, Calea standleyi, Calea tejadae, and Calea zacatechichi.
 17. Thepharmaceutical composition, according to claim 7, which comprises acompound having any one of structural formulae (1) to (7):

18 The food composition, according to claim 9, wherein said Calea isselected from the group consisting of Calea urticifolia, Caleapinnatifida, Calea uniflora, Calea axillaris, Calea insignis, Caleaintegrifolia, Calea nelsonii, Calea peduncularis, Calea pringlei, Caleapurpusii, Calea sabazioides, Calea savannarum, Calea scabra, Caleasororia, Calea standleyi, Calea tejadae, and Calea zacatechichi.
 19. Thefood composition, according to claim 9, which comprises a compoundhaving any one of structural formulae (1) to (7):